Peripheral mechanisms of tolerance and immunoregulation are essential to prevent the damage potentially caused by autoreactive T cells that escape thymic tolerance induction. Among these, Treg cells expressing the FoxP3 transcription factor are the best characterized, with their ability to suppress immune and autoimmune responses, and their exciting potential for human therapy. They represent a constant proportion of CD4+ cells in lymphoid organs of a given individual, but vary markedly between individuals in a species. Beyond known roles for IL2 and TGF2, the mechanisms and genetic variation that control the selection and homeostatic balance of the Treg pool remain unclear. Following studies on the survival and homeostasis of conventional T cells in the previous cycle, we propose to build on our recent work that dissected the Treg transcriptional signature, established that the thymic differentiation and peripheral homeostasis of Treg cells are under independent genetic control, and showed that the extra-lymphoid localization of Treg cells are conditioned by TCR sub-repertoires. We propose to study: 1) Cellular mechanisms of homeostatic control of Treg cells. We will assess Treg population dynamics (thymic export, survival, expansion, conversion in lymphopenic environments, homeostasis in extra-lymphoid sites), exploiting the 5-fold differences in steady- state Treg frequencies between different inbred strains for correlative analyses. We will determine whether the genetic differences are Treg autonomous or imparted by their lymphoid or stromal environment, and how they condition overall Treg function. Single-cell sequencing of their TCR repertoire will test whether variations are quantitative, or also affect qualitatively repertoire composition. 2) Secondary conversion to Treg phenotype: significance and specificity. Neo-conversion of mature CD4+ lymphocytes to a FoxP3+ state can be achieved by chronic antigen stimulation, lymphopenia-driven homeostatic expansion in vivo, or IL2+GF2 in vitro. Secondary Treg differentiation may be important for tolerance to self or to gut flora, but this speculation is conjectural, with no data on the true impact of conversion on Treg pools. Detailed population kinetic analyses with traceable FoxP3-GFP will address the extent, timing, and location of conversion. We will explore the contribution of Retinoic Acid in this process by analyzing Treg homeostasis in mice deficient in the key RAR1 receptor, in T or stromal cells. In a reversion of the usual experimental logic, we will use single-cell TCR sequencing and construction of retrogenic mice to test, not whether a given cell can be induced to convert, but which TCR specificities can lead Tconv cells to convert. 3) AKT and the molecular determinism of Treg cell homeostasis. We will complement these cellular studies by investigating the molecular determinants of Treg selection and conversion. We have shown that active AKT inhibits TGF2-induced conversion as well as thymic selection of Treg cells, an effect countered by rapamycin which affects a large segment of the Treg transcriptional signature. We will explore the role played by AKT in the homeostatic control of Treg populations and in vivo neo-conversion from mature Tconv cells, and the relationship between AKT signaling and the main cytokine mediators of Treg homeostasis, IL-2 and IL-6. Using RNAi knockdown and biochemical approaches, we will explore the pathways that connect AKT and FoxP3 expression, the upstream activators of AKT, and the downstream target(s) that are phosphorylated by AKT to mediate this phenomenon. . Public Health Relevance: These experiments will explore the cellular mechanisms and regulatory pathways that lead to variations in the differentiation and homeostasis of FoxP3+ T regulatory cells. The results should lead to a better understanding of this population that is key to immune and autoimmune regulation.